| PART ONE:The expression and clinical significances of 2-methoxyestradiol in early-onset preeclampsiaObjective To study the expression of 2-methoxyestradiol(2-ME)in the serum of patients with early-onset preeclampsia and its correlation with the clinical severity of the disease.Methods The pregnant women were selected from those who were examined and delivered in Xianning Central Hospital with gestational weeks more than 28 weeks and less than 34 weeks,singleton pregnancy,and diagnosed as preeclampsia(PE)during the period from March 2014 to March 2017,of which 25 cases with early-onset mild preeclampsia patients(EOMPE group),25 cases with early-onset severe preeclampsia patients(EOSPE group);25 cases of healthy pregnant women were randomly selected as the normal control group(NP group),who were singleton pregnancy,gestational weeks less than 34 weeks and premature birth.The related clinical data were collected for a statistical analysis,such as the age of the patient,gestational weeks,the history of gravidity and parity,BMI,systolic blood pressure,diastolic blood pressure,blood fat(triglyceride,cholesterol,low density lipoprotein),24-hour proteinuria,renal function(uric acid,creatinine,urea nitrogen),lactic dehydrogenase,D-dimer,expectant treatment time,neonate birth weight,Apgar scores,etc..The pregnancy serum 2-ME,soluble vascular endothelial growth factor receptor-1(sFlt-1),placental growth factor(PIGF)and endothelin-1 levels(ET-1)of each group were detected by enzyme linked immunosorbent assay(ELISA);the nitric oxide(NO)content of the pregnancy serum were measured by the method of nitrate reductase for each group.The correlation between the serum 2-ME and clinical indicators of EOSPE group was analyzed.What's more,the correlation between the serum 2-ME and the levels of sFlt-1,PIGF,ET-1,and NO of EOSPE group was also analyzed.Through the collection of placenta in NP group and EOSPE group to detect the expression of catecholamine-o-methyltransferase(COMT)in placenta by the immunohistochemical method,to measure the expression levels of COMT mRNA and protein in placenta by the methods of RT-PCR and Western Blot,and to make a statistical analysis.Results1.There were no significant differences in age,the history of gravidity and parity,gestational weeks,and pre-pregnancy body mass index of three groups(P>0.05);the pairwise comparisons of systolic blood pressure,diastolic blood pressure,body mass index of blood-drawing day,blood uric acid,and serum creatinine showed there were statistically significant(P<0.05).There were significant differences on urea nitrogen,triglyceride,total cholesterol,low density lipoprotein,lactic dehydrogenase,and D-dimer of pregnancy serum between the EOSPE group and NP group(P<0.05),while there were no significant differences on urea nitrogen,triglyceride,and lactic dehydrogenase of pregnancy serum between.the EOMPE group and NP group(P>0.05).There were no significant differences on gestational age and expectant treatment time between the EOSPE group and NP group(P>0.05).The 24-hour proteinuria of pregnant women in the EOSPE group was significantly higher than those in the EOMPE group,the neonatal weight and Apgar scores in the EOSPE group were lower than those in the NP group,and the differences were statistically significant(P<0.05).2.The 2-ME level of pregnancy serum in EOMPE group was(645.2± 177.2)pg/ml,the EOSPE group was(525.3± 172.6)pg/ml,and the NP group was(705.9± 157.4)pg/ml.The 2-ME level of pregnancy serum in EOSPE group was lower than those in EOMPE group and NP group,and the differences were statistically significant(P<0.05).However,there were no significant differences on the 2-ME level of pregnancy serum between the EOMPE group and the NP group(P=0.209).3.The sFlt-1 level of pregnancy serum in EOMPE group was(3413.4±619.7)pg/ml,the EOSPE group was(5199.2±584.0)pg/ml,and the NP group was(2940.0±465.4)pg/ml,pairwise comparisons were statistically significant(P<0.05).The sFlt-1 level of pregnancy serum in the preeclampsia group was significantly higher than those in the normal control group,and the increasing was obvious with the aggravation of the disease.4.The PIGF level of pregnancy serum in EOMPE group was(81.5±17.0)pg/ml,the EOSPE group was(77.4±23.5)pg/ml,and the NP group was(110.2±45.0)pg/ml;the PIGF level of pregnancy serum in EOSPE group was significantly lower than those in NP group(P<0.01),the PIGF level of pregnancy serum in EOMPE group was also lower than those in NP group(P<0.05),but there were no significant differences between the EOSPE group and EOMPE group(P=0.861).5.For the pregnancy serum of EOMPE group,the ET-1 level was(49.7±11.0)pg/ml,the NO level was(63.6±17.9)umol/l;for the pregnancy serum of EOSPE group,the ET-I level was(67.6± 11.2)pg/ml,the NO level was(48.1 ±9.3)umol/1;for the pregnancy serum of NP group,the ET-1 level was(32.5±7.2)pg/ml,the NO level was(74.7±21.9)umol/1,and pairwise comparisons were statistically significant(P<0.05).The ET-1 level of pregnancy serum in the preeclampsia group was higher than those in the normal control group,while the NO level decreased,and the increasing of the ET-1 level was obvious with the aggravation of the disease.6.Using the immunohistochemical method to detect the expression of COMT in placenta of the early-onset severe preeclampsia group and the normal control group,and the results showed that there was the expression of COMT in the placenta tissue of two groups,but there were no significant differences(P>0.05).The relative expression of COMT mRNA in placenta tissue of the EOSPE group was(0.447±0.152),and the NP group was(0.467±0.151),so there were no significant differences between the two groups(P=0.678);there were also no significant differences(P=0.422)on the expression of COMT protein in the placenta tissue between the EOSPE group(0.8916±0.1020)and the NP group(0.9240±0.1587).7.There was a negative correlation between the 2-ME level and systolic blood pressure,low density lipoprotein,triglyceride,plasma creatinine,urea nitrogenand 24-hour proteinuria in the early-onset severe preeclampsia group;there was a positive correlation between the 2-ME level and the expectant treatment time.The correlation coefficient R values were-0.648(P<0.01),-0.620(P<0.01),-0.649(P<0.01),-0.615(P<0.01),-0.524(P<0.01),-0.587(P<0.01),and 0.694(P>0.01).8.There was a negative correlation between the 2-ME level and the sFlt-1 level in the early-onset severe preeclampsia group;there was a positive correlation between the 2-ME level and PIGF level,NO level.The correlation coefficient R values were-0.645(P<0.01),0.511(P<0.01),0.853(P<0.01).However,there was no correlation between the 2-ME level and ET-1 level(P=0.146).ConclusionsThe serum 2-ME level of the early-onset preeclampsia pregnant women was lower than that of normal pregnant women.Compared with the normal control group,the expression of COMT in placenta of the early-onset severe preeclampsia pregnant women was not changed.The expression level of 2-ME in the serum of the early-onset severe preeclampsia pregnant women was related to the severity of the disease.PART TWO:The effect of the serum of early-onset severe preeclampsia on HUVECs and its cell injuryObjective To explore the effect of the serum on the proliferation,apoptosis and endocrine function of human umbilical vein endothelial cells in patients with early-onset severe preeclampsia,in order to successfully construct the endothelial cells injury model of preeclampsia.Methods The human umbilical vein endothelial cells(HUVECs)were cultured and grouped as follows:10%healthy pregnant women of preterm labor with serum stimulation(normal control group),10%patients of early-onset severe preeclampsia with serum stimulation(PE group),and the blank control group(without any serum stimulation).After the serum-free culture medium was cultured for 24 hours,(1)The morphological changes of HUVECs were observed under the inverted microscope.(2)The method of CCK-8 was used to detect cells proliferation ability.(3)The double staining of Annexin V-FITC/PI and flow cytometer were used to detect the effects of the serum on cells apoptosis in each group.(4)The enzyme linked immunosorbent assay was used to analyze and measure the endothelin(ET-1)content in cell culture supernate.(5)The nitrate reductase method was used to measure the NO content in culture supernate.All the results were analyzed statistically.Results(I)Under the inverted microscope we can see that:the intercellular space of PE group was larger,parts of cells shrank and became round,the intercellular space widened,the cytoplasm reduced,and the cell death and abscission can be found in local areas.The HUVECs of the normal control group and the blank control group with uniform size,and had abundant cytoplasm.(2)The cell proliferative ability of each group was detected by CCK8 kit and represented with the OD value.Compared with the normal control group(0.8966±0.0821)and the blank control group(0.9140±0.1201),the cell proliferative ability of PE group(0.5118±0.0782)decreased significantly,and the differences were statistically significant(P<0.01).(3)The apoptosis rate of each group was detected by Annexin V-FITC/PI kit.Compared with the normal control group(7.88±1.58)%and the blank control group(7.34±1.78)%,the cell apoptosis rate of PE group(16.57±2.257.56±1.25)%increased obviously,and the differences were statistically significant(P<0.01).(4)Compared with the normal control group(0.2423±0.0455)pg/ml and the blank control group(0.2450±0.0781)pg/ml,the ET-1 content(0.5430±0.0952)pg/ml in cell culture supernate of PE group increased obviously,and the differences were statistically significant(P<0.01).(5)Compared with the normal control group(27.07±2.78)umol/1 and the blank control group(27.12± 1.98)umol/1,the NO concentration(18.18±2.45)umol/l in cell culture supernate of PE group decreased obviously,and there were significant differences(P<0.01).Conclusions The serum of patients with early-onset severe preeclampsia can decrease the proliferation rate of HUVECs,lead to apoptosis,secretion of ET-1 and decrease the content of NO significantly.It can induce the injury of HUVECs.We successfully construct the endothelial cell injury model of preeclampsia.PART THREE:Protective effect and preliminary mechanism of 2-ME on injury of HUVECs caused by serum in patients with early-onset severe preeclampsiaObjective To determine the appropriate concentration and time of 2-ME acting on HUVECs;to study the protective effects of 2-ME on the injury of human umbilical vein endothelial cells caused by serum in patients with early-onset severe preeclampsia,and to explore its possible preliminary mechanisms.Methods(1)10%healthy pregnant women of preterm labor with serum stimulation(normal control group),10%patients of early-onset severe preeclampsia with serum stimulation(PE group),HUVECs were cultured in 2-ME with five concentration gradients(0,1,2,4,8?M)of each group and the blank control group(without any serum stimulation)was also established.The cell viability was detected by CCK-8 kit after HUVECs were cultured for 12,24,48 hours in each group.Each group was equipped with 3 double pores and the experiment was repeated six times.The suitable concentration and action time of 2-ME dosing were screened.(2)The morphological and structural changes of HUVECs were observed under the inverted microscope.(3)The HUVECs were cultured and grouped as follows:10%healthy pregnant women of preterm labor with serum stimulation(NC group),10%healthy pregnant women of preterm labor with serum stimulation +2-ME(NC+2-ME group),10%patients of early-onset severe preeclampsia with serum stimulation(PE group),10%patients of early-onset severe preeclampsia with serum stimulation +2-ME(PE+2-ME group),and the blank control group(without any serum stimulation).After the HUVECs were cultured,the endothelin-1(ET-1)content in cell culture supernate was analyzed and measured by enzyme linked immunosorbent assay and the NO content in cell culture supernate was measured by nitrate reductase method.(4)The transcriptional level changes of VEGF,HIF-1? and sFlt-1 were detected by RT-PCR after treatment with 2-ME.(5)The secretion level changes of sFlt-1 in cell culture supernate were detected by enzyme linked immunosorbent assay(ELISA)after treatment with 2-ME,and the protein expression changes of HIF-1? and VEGF were detected by Western Blot after treatment with 2-ME.Results(1)There were no differences on cell viability between the blank control group and the normal control group.Compared with the normal control group,the viability of HUVECs in PE group decreased obviously.The viability of HUVECs presented an increasing trend with the rising of 2-ME concentration after treatment with 2-ME.Finally,the concentration of 2-ME was 4?M and the action time was 24 hours.(2)HUVECs of NC group and the blank control groupwere cultured with uniform size,abundant cytoplasm,polygon or triangle,and arranged with the shape of single-layer inlaid paving stones.The cells treated in PE group presented typical cell injury,the cytoplasm reduced,the intercellular space was larger,parts of cells shrank and became round;and the cell death and abscission can be found in local areas.In PE+2-ME group,the cell injury of HUVECs was repaired partially,which can be observed under the microscope.The cell morphology was polygon or oval,arranged closely,and the cell boundary was clear.(3)The ET-1 content in culture supernate was measured by ELISA after treatment with 2-ME.Compared with NP group(0.242±0.010)pg/ml,the ET-1 content in culture supernate increased obviously in PE group(0.543±0.058)pg/ml,which suggested that the injury effects of severe preeclampsia on cells.The ET-1 content(0.331 ±0.055)pg/ml decreased obviously after using 2-ME intervention.(4)The changes of NO content in culture supernate were measured by nitrate reductase method after treatment with 2-ME.Compared with NC group(27.12± 1.58)umol/l,the NO content in culture supernate decreased in PE group(18.18±0.38)umol/l.Compared with PE group,the NO content increased obviously in PE+2-ME group(22.90±1.19)umol/1.All of these results showed that the groups treated with 2-ME can reverse this change,which led to the recovery of NO content in supernate.(5)The transcriptional level changes of VEGF,HIF-la and sFlt-1 were detected by RT-PCR after treatment with 2-ME.Compared with NC group(0.98±0.07),the transcriptional level of VEGF reduced obviously in PE group((0.57±0.13),and the difference was statistically significant(P<0.05);but the transcriptional level of VEGF(0.84±0.06)increased after treatment with 2-ME(P<0.05),which suggested that 2-ME can reverse the transcriptional level of VEGF.Compared with NC group(HIF-1?a(1.02±0.04),sFIt-1(0.95±0.05)),the transcriptional level of HIF-1?(1.43±0.06)and sFIt-1(1.64±0.10)increased obviously in PE group,and the difference was statistically significant(P<0.05);but the transcriptional level of HIF-1?(1.11±0.05)and sFIt-1(0.86±0.09)reduced obviously(P<0.05)after treatment with 2-ME,which suggested that 2-ME can improve the transcriptional level of HIF-1? and sFlt-1.(6)The secretion level changes of sFlt-1 in cell culture supernate were detected by ELISA after treatment with 2-ME.Compared with NC group(454±7)pg/ml,the sFlt protein content of cell secretion increased in PE group(540±38)pg/ml,and there was a significant difference(P<0.05);but the sFlt-1 protein level(493±16)pg/ml reduced obviously(P<0.05)after treatment with 2-ME.The protein expression changes of VEGF and HIF-1? were detected by Western Blot after treatment with 2-ME.Compared with NC group(61.9± 1.0)%,the protein expression of VEGF reduced in PE group(37.3±4.0)%,and there was a significant difference(P<0.05);but the protein expression(52.4±5.9)%of VEGF increased after treatment with 2-ME(P<0.05).Compared with NC group(44.2± 1.0)%,the protein expression of HIF-1? increased in PE group(92.6±4.0)%,and there was a significant difference(P<0.05);but the protein level of HIF-1?(65.9±4.5)%reduced obviously after treatment with 2-ME(P<0.05).All of the results suggested that 2-ME can reverse the protein expression of VEGF and reduce the protein expression of HIF-la and sFlt-1.Conclusions 2-ME can improve HUVEC cells dysfunction by increasing the proliferation rate of HUVEC cells,reducing the release of endothelin-1(ET-1)and increasing NO synthesis.Its mechanism can play a protective role for HUVEC cells may be through reducing the expression of HIF-la,inhibiting the secretion of sFlt-1 and promoting the expression of VEGF. |
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